1,721,005 research outputs found
A novel thermophilic Glycosynthase that effects branching glycosylation
No full textA novel thermophilic glycosynthase that effects branching glycosylation has been obtained by mutation of the nucleo- phile in the active site of the glycosidase from Sulfolobus solfataricus. Two methods for the use of this mutant are reported
Highly productive autocondensation and transglycosylation reactions with Sulfolobus solfataricus glycosynthase
No full textTransglycosylation reactions (autocondensation of the substrate or transfer of the glycon donor moiety to different acceptors) with the hyperthermophilic glycosynthase from Sulfolobus solfataricus acting in dilute sodium formate buffer at pH 4.0 are reported; the use of 4-nitrophenyl beta-glucopyranoside as both donor and acceptor in the self-transfer reaction and a highly productive reaction with 1.1 M 2-nitrophenyl beta-glucopyranoside were possible. Interesting effects, governed by the anomeric configuration and lipophilicity of heteroacceptors, on the regioselectivity and yield of reactions were found for the first time with this enzyme and are discussed. The results demonstrate the unexplored synthetic potential of this glycosynthase; the tuning of the reaction conditions and the choice of different donors/acceptors can lead to products of applicative interest
Oligosaccharide synthesis by glycosynthases
No full textOligosaccharide synthesis is becoming increasingly important to the pharmaceutical industry because these biomolecules have potential application as therapeutics. Enzymatic synthesis of oligosaccharides includes both glycosyl transferases and glycosidases. The main advantage of this approach compared with traditional chemical synthesis is the regio- and stereo-selectivity that can be achieved without the need for protecting functional groups. However, the limited availability of glycosyl transferases, the high cost of their substrates, and the poor yields of the synthetic reactions per- formed by the glycosidases hamper their use in the large-scale production of oligosaccharides. The recent advent of glycosynthases – specifically mutated glyco- sidases that efficiently synthesize oligosaccharides but do not hydrolyse them – represents a promising sol- ution to these problems. Here, we review the most recent developments in the glycosynthase approach
Restoration of the activity of active-site mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles
No full textThe beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed
Glycosynthase-Catalysed syntheses at pH below neutrality
No full textThe use of the new glycosynthase Ta-b-GlyE386G and of the already known Ss-b-GlyE387G for the synthesis of inter- esting 4-methylumbelliferyl disaccharides and for the galactosylation of a- and b-xylosides of 4-penten-1-ol is reported. The results show satisfactory yields of reaction in presence of low excesses of acceptors and demonstrated that the high activity of these enzymes at pH below neutrality is applicable in the transfer of glucose as well as of galactose from the preferred 2-NP-based donors
Genome stability: recent insights in the topoisomerase reverse gyrase and thermophilic DNA alkyltransferase
No full textRepair and defence of genome integrity from endogenous and environmental hazard is a primary need for all organisms. Natural selection has driven the evolution of multiple cell pathways to deal with different DNA damaging agents. Failure of such processes can hamper cell functions and induce inheritable mutations, which in humans may cause cancerogenicity or certain genetic syndromes, and ultimately cell death. A special case is that of hyperthermophilic bacteria and archaea, flourishing at temperatures higher than 80 °C, conditions that favor genome instability and thus call for specific, highly efficient or peculiar mechanisms to keep their genome intact and functional. Over the last few years, numerous studies have been performed on the activity, function, regulation, physical and functional interaction of enzymes and proteins from hyperthermophilic microorganisms that are able to bind, repair, bypass damaged DNA, or modify its structure or conformation. The present review is focused on two enzymes that act on DNA catalyzing unique reactions: reverse gyrase and DNA alkyltransferase. Although both enzymes belong to evolutionary highly conserved protein families present in organisms of the three domains (Eucarya, Bacteria and Archaea), recently characterized members from hyperthermophilic archaea show both common and peculiar features
Positive supercoiling in thermophiles and mesophiles: of the good and evil
No full textDNA supercoiling plays essential role in maintaining proper chromosome structure, as well as the equilibrium between genome dynamics and stability under specific physicochemical and physiological conditions. In mesophilic organisms, DNA is negatively supercoiled and, until recently, positive supercoiling was considered a peculiar mark of (hyper)thermophilic archaea needed to survive high temperatures. However, several lines of evidence suggest that negative and positive supercoiling might coexist in both (hyper)thermophilic and mesophilic organisms, raising the possibility that positive supercoiling might serve as a regulator of various cellular events, such as chromosome condensation, gene expression, mitosis, sister chromatid cohesion, centromere identity and telomere homoeostasis.</jats:p
O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability
Full text linkThe genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as proteintags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications
Engineering the stability and the activity of a glycoside hydrolase
No full textGlycosidases, the enzymes responsible in nature for the catabolism of carbohydrates, are well-studied catalysts widely used in industrial biotransformations and oligosaccharide synthesis, which are also attractive targets for drug development. Glycosidases from hyperthermophilic organisms (thriving at temperatures > 85 °C) are also interesting models to understand the molecular basis of protein stability and to produce robust tools for industrial applications. Here, we review the results obtained in the last two decades by our group on a β-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus. Our findings will be presented in the general context of the stability of proteins from hyperthermophiles and of the chemo-enzymatic synthesis of oligosaccharides
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